PROJECT SUMMARY Aberrant signaling through receptor tyrosine kinases of ERBB family members plays a key role in the etiology of breast cancer. Approximately 20-25% of invasive breast cancers exhibit an amplification of the ERBB2 locus or a constitutive activation of the receptor tyrosine kinase encoded by this gene. There is a substantial body of experimental and clinical data that shows that ERBB2-overexpressing breast cancer cells have an elevated metastatic potential. Despite initial response to targeted therapies against ERBB2 using trastuzumab and pertuzumab, the majority of patients eventually relapse and succumb to metastatic disease. Therefore, novel therapeutic strategies are needed to prevent and treat this aggressive breast cancer subtype. As a component of the endocytic machinery, the protein encoded by the Tumor Susceptibility Gene 101 (TSG101) is crucial for the intra-cellular trafficking, sorting, and lysosomal degradation of ubiquitinated cargo proteins including ERBB2 and other receptor tyrosine kinases. TSG101 is a central node in trafficking as it is able to simultaneously bind cargo proteins as well as PTAP amino acid motif-containing regulatory proteins and ubiquitin ligases that modify the sorting and trafficking of cargo complexes. TSG101 itself possesses an intrinsic PTAP motif near its C-terminal end, and we gathered preliminary evidence that this domain can associate with the PTAP binding groove at the N-terminus of TSG101. Moreover, we can demonstrate that mutating the intrinsic PTAP motif into an ATAA amino acid sequence and thereby blocking the intramolecular binding modality leads to a very significant decrease in the steady-state level of TSG101 and a simultaneous decline in the expression of its cargo proteins ERBB2, EGFR, and IGF-R1. The immediate objectives of this exploratory project are to extend the preliminary findings from in vitro studies and to develop a TSG101-ATAA knockin mouse model using the CRISPR/Cas9-based gene editing approach to firmly establish that disrupting the intramolecular association of the intrinsic PTAP domain with its binding grove increases the turnover and reduces the expression of TSG101 in vivo. We will then apply this mutant TSG101-ATAA knockin model to assess whether lowering the steady-state level of TSG101 will be sufficient to co-downregulate oncogenic ERBB2 and prevent the onset and progression of ERBB2-indcued mammary cancer in transgenic mice. Accelerating the turnover of TSG101 and its cargos represents a novel approach to prevent and treat ERBB2- positive breast cancer, and the collective results from this exploratory project will provide strong in vivo evidence for the development of a new class of therapeutics that target specific intramolecular associations within TSG101.